NOTCH receptors 1-4 are transmembrane receptor proteins that signal through a pathway that relies on regulated proteolysis. Following ligand-binding, the receptor is sequentially: i) cleaved extracellularly by metalloproteases of the ADAM family (Brou, et al., Mol. Cell. 5:207-216 (2000); Mumm, et al. Mol Cell 5:197-206 (2000)); ii) mono-ubiquitinated on a lysine residue lying just internal to the transmembrane domain (Gupta-Rossi, et al., J. Cell Biol. 166:73-83 (2004)); iii) endocytosed (Gupta-Rossi, et al., J. Cell Biol. 166:73-83 (2004)), and iv) proteolytically cleaved by a gamma-secretase enzyme (De Strooper, et al., Nature 398:518-522 (1999)). This final step in the activation process permits the intracellular portion of NOTCH receptors to translocate to the cell nucleus where it interacts with transcription factors to alter gene activity. NOTCH receptor signaling appears to play an important role in the differentiation and proliferation of cells and in controlling apoptosis, three processes that are important with respect to neoplastic transformation (see U.S. Pat. No. 6,703,221).
The NOTCH-1 gene was discovered through its involvement in a (7; 9) chromosomal translocation found in fewer than 1% of T-cell acute lymphoblastic leukemias (T-ALLs) (Ellisen, et al., Cell 66:649-661 (1991)). NOTCH-1 is highly expressed in thymocytes (Ellisen, et al., Cell 66:649-661 (1991)), where it induces common lymphoid progenitors to adopt a T cell fate (Radtke, et al., Immunity 10:547-558). Subsequently, it promotes the assembly of pre-T cell receptor complexes which play a critical role in driving a proliferative burst that accompanies maturation of CD4−/CD8− thymocytes to the CD4+/CD8+ developmental stage (Wolfer, et al., Immunity 16:869-879 (2002)).
In its resting state, mature NOTCH-1 is a heterodimeric receptor comprised of a ligand-binding extracellular subunit (NEC) and a non-covalently associated transmembrane subunit (NTM) (Rand, et al., Mol. Cell. Biol. 20:1825-1835; Logeat, et al., Proc. Natl. Acad. Sci. USA 95:8108-8112 (1998)). NEC consists of a ligand-binding domain comprised of epidermal growth factor-like repeats, three iterated Lin 12/NOTCH repeats, and a conserved 103 amino acid sequence (hereafter termed HD, for heterodimerization domain) that is sufficient for association with the extracellular portion of NTM.
Physiologic activation of NOTCH receptors occurs when a ligand of the Delta-Serrate-Lag2 (DSL) family binds to the NEC subunit and initiates a cascade of successive proteolytic cleavages in the NTM subunit. The final cleavage, which is catalyzed by γ-secretase, a multiprotein complex containing presenilin-1 or -2, nicastrin, APH-1, and PEN-2 (Francis, et al., Dev. Cell 3:85-97 (2002); Kimberly, et al., Proc. Natl. Acad. Sci. USA 100:6382-6387 (2003)) releases the intracellular part of NTM (called intracellular NOTCH, or ICN) from the membrane, permitting it to translocate to the cell nucleus. There, it associates with the DNA-binding factor CSL and co-activators of the Mastermind family to form a short-lived transcriptional activation complex (Wallberg, et al., Mol. Cell. Biol. 22:7812-7819 (2002); Fryer, et al., Genes Dev. 16:1397-1411 (2002); Nam, et al., J. Biol. Chem. 278:21232-21239 (2003)) Degradation and turnover of the complex is apparently regulated by F-box factors of the SEL-10 family (Oberg, et al., J. Biol. Chem. 276:35847-35853 (2001)).
The (7; 9) translocation creates a NOTCH-T cell receptor β fusion gene that encodes N-terminally-deleted, constitutively active NOTCH-1 polypeptides similar to the ICN (Ellisen, et al., Cell 66:649-661 (1991); Aster, et al., Cold Spring Harb. Symp. Quant. Biol. 59:125-136 (1994); Das, et al., J. Biol. Chem., epublished May 3, 2004)) and these truncated and constitutively active forms of NOTCH-1 induce T-ALL in mouse models (Aster, et al., Mol. Cell. Biol. 20:7505-7515 (2000)). NOTCH-1 is also the site of frequent retroviral insertions that cooperate with the E2A-PBX1 and cMYC transgenes in multistep pathways leading to the development of murine T-ALL (Hoemann, et al., Mol. Cell. Biol. 20:3831-3842 (2000); Feldman, et al., Blood 96:1906-1913 (2000)). Further, NOTCH inhibitors cause a G0/G1 cell cycle arrest in cell lines derived from human and murine NOTCH-1-associated T-ALLs, indicating that NOTCH signaling is required for the sustained growth of these leukemias (Weng, et al., Mol. Cell. Biol. 23:655-664 (2003)).
The (7; 9) translocation has only been associated with a very small percentage of patients having T-ALL. The identification of additional mutations associated with NOTCH-1 would have important implications for the pathogenesis of this type of cancer. Although mutations in NOTCH-2, NOTCH-3, and NOTCH-4 have not been identified in human cancer, it is known that abnormal increases in function of these NOTCH receptors in other mammals can cause T-ALL (NOTCH-2 and -3, Bellavia, et al., Embo J. 19:3337-3348 (2000); Rohn, J. Virol. 70:8071-8080 (1996); Weng, et al., Mol. Cell. Biol. 23:655-664)) and breast cancer (NOTCH-4, Callahan and Rafat, J. Mammary Gland Biol Neoplasia 6:23-36 (2001)). Identification of novel mutations in human tumors should be useful diagnostically in helping to identify the presence of cancer and in identifying cancer cells that respond to inhibitors of NOTCH signaling, thereby making it possible to direct rational cancer treatment with NOTCH signaling pathway inhibitors.